Why does amplification efficiency decrease or plateau in the late cycles?

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Three limitations may contribute to the decrease in amplification efficiency:

1. Plateau occurs in PCR because of the progressive reduction in the efficiency of the primer-template complex formation due to product reannealing. The primers in PCR are always present at high excess and the kinetics of reannealing are determined by the molecule in the greatest concentration. Typically, total initial PCR primer concentrations are 2x10E-6 M. However, as the reaction proceeds, the DNA target concentration increases and the primer concentration decreases, albeit only slightly, to 1.4x10E-7 M to 2x10E-6 M, and it takes a longer time for the primer-template complex to form. If the product strands have a chance to reanneal to themselves, forming a very stable complex, the primers will not have a binding site for extension and doubling may not occur. This snap-back of product strands reannealing to themselves can occur in a few seconds (or less) at high DNA concentrations.

2. Primer concentration depletion. One cause of primer depletion is the formation of primer artifacts and other spurious, non-specific amplification products.

3. Insufficient enzyme concentration or polymerization time in late cycles. In a typical PCR amplification, 2.0 - 2.5 U or roughly 8x10E10 molecules of AmpliTaq™ DNA Polymerase are used. In plateau there are roughly 1x10E12 template copies leaving less than one molecule of AmpliTaq™ DNA Polymerase per primer-template complex. One way to overcome the limiting polymerase is to increase the extension time in the later cycles of the PCR amplification.

Answer Id: E1096

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What is the difference between the TaqMan™ Universal PCR Master Mix Cat. No. 4304437 and Cat. No. 4324018?

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Both part numbers for TaqMan™ Universal PCR Master Mix are supplied at a 2X concentration and contain the following components: AmpliTaq Gold™ DNA polymerase, dNTPs with dUTP, Passive Reference (ROX), and optimized buffer components. However, the part number 4304437 also contains AmpErase™ UNG and is not suitable to be used for One Step RT PCR.

Answer Id: E1581

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What master mix can I use with the TaqMan™ SNP Genotyping Assays?

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We recommend using either the TaqMan™ Universal Master Mix (I or II) or the TaqMan™ Genotyping Master Mix. The TaqMan™ Genotyping Master Mix has the advantage of proven performance with up to 3 days of pre- and post-PCR stability, allowing you to set up plates ahead of time or read the plates later (see the data here, https://tools.thermofisher.com/content/sfs/brochures/cms_039236.pdf).

Answer Id: E7523

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Why are miRNAs so important?

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MicroRNAs are believed to play an important role in post transcriptional gene regulation. A total of ~850 unique miRNAs have been discovered so far, including ~333 human miRNAs. They are relatively abundant, ranging from a few to as many as 50,000 molecules per cell. They account for approximately 1% of the predicted genes in animals and plants. The level of individual miRNAs varies dramatically across cell type and developmental stages. These differences in miRNA level are believed to be a key indicator of miRNA activity.

Recent research has implicated miRNAs in the following: cell development, differentiation, communication and cell death; DNA methylation and chromatin modification; metabolism; human cancer development; haematopoiesis; nervous system patterning; insulin secretion.

Answer Id: E2512

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What Applied Biosystems™ reagents and instruments can I use with TaqMan™ Gene Expression Assays?

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The TaqMan™ Gene Expression Assays are optimized to work with one of the TaqMan™ qPCR Master Mixes that include TaqMan™ Universal PCR Master Mix, No AmpErase™ UNG(P/N 4324018), TaqMan™ Universal PCR Master Mix with AmpErase UNG (P/N 4304437), TaqMan™ Universal PCR Master Mix II, No AmpErase™ UNG (P/N 4440040), TaqMan™ Universal PCR Master Mix II, with AmpErase™ UNG (P/N 4440038) or TaqMan™ Gene Expression Master Mix (P/N 4369016) and with your cDNA. It is recommended to use the High Capacity cDNA Archive Kit (P/N 4322171) for converting your RNA to cDNA. TaqMan™ Gene Expression Assays are capable of being run on all Applied Biosystems™ Real-Time PCR Systems models including 7000, 7300, 7500, 7500 Fast, 7700, 7900HT, StepOne™, StepOnePlus™ and ViiA™ 7 Instruments.

Answer Id: E1834

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What is mechanism of the TaqMan™ MicroRNA Assays?

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There are 2 steps involved in using the TaqMan™ MicroRNA Assays:

1. RT step: total RNA to cDNA using miRNA-specific RT primers from the TaqMan™ MicroRNA Assays and the TaqMan™ MicroRNA Reverse Transcription Kit (P/N 4366596, 200 reactions or 4366597, 1000 reactions)

2. PCR step: cDNA to PCR products using primers and probe from the TaqMan MicroRNA Assays together with TaqMan™ Universal PCR Master Mix No AmpErase™ UNG (P/N 4324018).

Answer Id: E2516

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How do the miRNA assays correspond to miRBase?

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Individual TaqMan™ miRNA Assays are designed to target specific mature miRNA sequences. The mature sequences correspond to those contained in miRBase. Often several precursor miRNAs with different names and sometimes across several species will produce the identical mature miRNA sequence. Thus, one particular assay may be valid for use across several species. The Assay Index File provides the cross species key for TaqMan™ MicroRNA Assays.

Answer Id: E2514

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What does it mean when a miRNA target entry has been removed (obsoleted) from miRBase?

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miRBase continues to re-annotate the miRNA sequences as more information becomes available. A sequence that once mapped to a given species (e.g.: human) may no longer exist, so it is removed from miRBase. Accordingly, the original assay for that sequence no longer maps to an annotated miRNA, and as a result the annotation fields on the search results page on the Life Technologies portal are empty. An example of this is Assay “hsa-miR-505” (Assay ID 001049). miRBase continues to re-annotate the miRNA sequences as more information becomes available. A sequence that once mapped to a given species (e.g.: human) may no longer exist, so it is removed from miRBase. Accordingly, the original assay for that sequence no longer maps to an annotated miRNA, and as a result the annotation fields on the search results page on the Life Technologies™ portal are empty. An example of this is Assay “hsa-miR-505” (Assay ID 001049).

Answer Id: E2522

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If I inadvertently put the TaqMan™ Universal Master Mix (Cat. No. 4304437) in the freezer at -20 degrees C, can I still use it for my experiments?

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Answer

Although it is not recommend to freeze the Universal Master Mix, testing has shown that if it is frozen and then thawed, you should not experience any difficulties. If the Universal Master Mix is frozen, make sure that it is thoroughly thawed and mixed prior to use, followed by proper storage at 4 degrees C.

Answer Id: E1550

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What is the TaqMan™ MicroRNA Assay?

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A TaqMan™ MicroRNA Assay has two components: an RT reaction containing a miRNA specific stem-loop reverse-transcription primer and a separate specific TaqMan™ miRNA Assay. The RT stem-loop primer provides the specificity for the mature miRNA target; it does not detect its precursor. The formation of a RT primer/mature miRNA-chimera extends the length of the 5’ end of the miRNA. The longer RT product provides a miRNA specific cDNA template amenable to the TaqMan™ assay design.

Answer Id: E2519

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What are miRNAs?

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Answer

MicroRNAs (miRNAs) are endogenous non-coding RNAs, about 22 nucleotides in length, that play an important role in post-transcriptional gene regulation in animals and plants by targeting messenger RNA (mRNA) for degradation or translational repression. The miRNA precursors are transcribed from individual genes but are not translated into proteins. The primary transcript is processed in the nucleus to give one or more hairpin precursors that is exported to the cytoplasm. The mature miRNA is excised from the hairpin precursor. The mature miRNA is the biologically active form and regulates the expression of mRNA transcripts by binding to complementary sites on target mRNAs and inhibiting translation or, when there is perfect complementarity to the target sequence, inducing mRNA cleavage. In animals, translational repression, not transcript degradation, is the dominant miRNA mode of action.

Answer Id: E2510

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Can dU-containing DNA produced by amplification with dUTP be cut by restriction enzymes?

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You will need to confirm with the manufacturer of the restriction enzyme you are using to find out whether or not it can recognize dUTP-containing DNA.

Answer Id: E1411

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Can I use TaqMan™ 2X Universal PCR Master Mix (including AmpErase™ UNG with TaqMan™ MicroRNA Assays?

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TaqMan™ MicroRNA Assays protocol (Cat. No. 4364031) recommends TaqMan™ 2X Universal PCR Master Mix, No AmpErase™ UNG (Cat. No. 4324018). However, all TaqMan™ MicroRNA Assays can be used with TaqMan™ 2X Universal PCR Master Mix, with AmpErase™ UNG (Cat. No. 4304437).

Answer Id: E2770

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Can I check the end product of a TaqMan™ Gene Expression Assay by running an agarose gel?

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Yes, you can check the end product on an agarose gel. If you use the TaqMan™ Universal PCR Master Mix (including AmpErase™ UNG, Cat. No. 4304437), we recommend running the gel immediately after the PCR, since the trace amount of AmpErase™ UNG may digest the end product that contains double-stranded DNA with dUTP incorporated. The average length of amplicon ranges from 50-150 bp. The exact amplicon length is indicated on the detailed page of each assay on our website. You can get to that page by clicking on the Assay ID; the amplicon information is listed in the section "Assay Details".

Answer Id: E2464

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What is the purpose of the RNase P Detection Reagents Kit (Cat. No. 4316831), and what is provided in the kit?

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Answer

RNase P is a single-copy gene encoding the RNA moiety for the RNase P enzyme, and it is an excellent gene to use as a reference/control for Copy Number Variation (CNV) assays. The TaqMan™ RNase P Detection Reagents Kit contains a 20X mix of primers and probe (FAM™ dye-labeled) that will detect and quantitate genomic copies of the human RNase P gene. The primers and probe are designed according to TaqMan™ chemistry guidelines for quantitation, and utilize the universal thermal cycling parameters. This kit is used to perform a 5' nuclease assay with TaqMan™ Universal PCR Master Mix (Cat. No. 4304437) or the TaqMan™ Core PCR Reagents Kit (Cat. No. N808-0228) with genomic, plasmid or complementary DNA (cDNA) templates.

The complete components of the TaqMan™ RNase P Detection Reagents Kit, Cat. No. 4316831, are: 20X RNase P Primer-Probe (FAM™/TAMRA™ probe) Mix, 1 tube Human Genomic Control DNA, 10 ng/ul (100 ul), and the Product Insert with an overview and procedure for the use of the kit.

Answer Id: E1421

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